The long-range goals of our research are to understand the molecular mechanisms of transcription and replication of negative (-) strand RNA viruses. We have established a mammalian expression system using cloned genes to synthesize the Sendai virus L, P, and NP proteins which are necessary and sufficient for Sendai virus RNA synthesis in vitro. Two protein complexes, P-L and NP-P, form the essential units for RNA synthesis, where P-L is the viral RNA dependent RNA polymerase and NP-P is the substrate for NP encapsidation during RNA replication. We propose to study the structure and function of the Sendai P protein in Sendai virus RNA synthesis. We have located the L binding domain on P protein and propose to do site-directed mutagenesis in this domain of P to identify the amino acids critical for binding and RNA synthesis. Similarly, we will fine-map the two NP binding sites on the P protein and characterize the P- P oligomerization site. Phosphatase inhibitors and mutagenesis of conserved serines in the P protein will be used to identify phosphorylated residues required for Sendai RNA synthesis. We propose to study the structure and function of the L protein subunit of negative strand virus RNA polymerases by several approaches. The p binding site on the Sendai virus L protein will be mapped with the two-hybrid system in yeast in conjunction with site-directed mutagenesis of the L gene to define the contact points in the Sendai P-L complex. We will screen for host proteins interacting with the polymerase using the two hybrid system. Mutagenesis should create Sendai L mutants to define defects in RNA synthesis in vivo and in vitro. We will characterize a putative RNA binding region in conserved domain II of the Sendai virus L protein by binding assays and mutagenesis. We propose to identify the methyltransferase domain on the VSV L gene by sequencing wild type and methyltransferase defective VSV in conjunction with UV crosslinking the methylation substrate S-adenosyl- methionine to the VSV L protein. We propose to study the structure and function of the Sendai virus NP protein in RNA replication. Site-directed mutagenesis on the Sendai virus NP gene will be used to define the NP-P and NP-NP interactions and analyze the effects on RNA replication. We will test the isolated conserved region of the NP protein for RNA and protein binding. Cis sequences on the RNA template which are required for replication will be defined with the use of a cDNA clone for a defective interfering particle. We will study the mechanisms by which the non- structural C proteins inhibit Sendai virus transcription, while the non- structural V protein specifically inhibits RNA replication by mutagenesis and screening for interacting viral and host cellular proteins.